Lysine acetylation in the lumen of the ER: A novel and essential function under the control of the...

Biochimica et Biophysica Acta 1833 (2013) 686–697

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Review

Lysine acetylation in the lumen of the ER: A novel and essential function under thecontrol of the UPR

Mariana Pehar a,1, Luigi Puglielli a,b,⁎a Department of Medicine, University of Wisconsin-Madison, VA Medical Center, 2500 Overlook Terrace, Madison, WI 53705, USAb Geriatric Research Education Clinical Center, VA Medical Center, 2500 Overlook Terrace, Madison, WI 53705, USA

⁎ Corresponding author at: Department of Medicine, Unand GRECC-VAH, 2500 Overlook Terrace, Madison, WI1901x11569; fax: +1 608 280 7291.

E-mail address: [email protected] (L. Puglielli)1 Present Address: Department of Cell and Molecular

tal Therapeutics, Medical University of South Carolina, C

0167-4889/$ – see front matter © 2012 Elsevier B.V. Alhttp://dx.doi.org/10.1016/j.bbamcr.2012.12.004

a b s t r a c t
a r t i c l e i n f o
Article history:Received 8 October 2012Received in revised form 3 December 2012Accepted 5 December 2012Available online 13 December 2012

Keywords:Endoplasmic reticulumLysine acetylationATaseAT-1PCSK9/NARC-1Autophagy/ERAD(II)

The Nε-amino group of lysine residues can be transiently modified by the addition of an acetyl group. Recog-nized functions of Nε-lysine acetylation include regulation of activity, molecular stabilization and conforma-tional assembly of a protein. For more than forty years lysine acetylation was thought to occur only in thecytosol and nucleus. Targets included cytoskeletal-associated proteins as well as transcription factors, histoneproteins and proteins involved in DNA recombination and repair. However, in 2007 we reported that a type Imembrane protein involved in the pathogenesis of Alzheimer's disease was transiently acetylated on the εamino group of seven lysine residues while transiting along the secretory pathway. Surprisingly, the acetyla-tion occurred in the lumen of the endoplasmic reticulum (ER) forcing us to reconsider old paradigms. Indeed,if lysine acetylation can occur in the lumen of the ER, then all the essential biochemical elements of the reac-tion must be available in the lumen of the organelle. Follow-up studies revealed the existence of ER-basedacetyl-CoA:lysine acetyltransferases as well as a membrane transporter that translocates acetyl-CoA fromthe cytosol into the ER lumen. Large-scale proteomics showed that the list of substrates of the ER-based acet-ylation machinery includes both transiting and resident proteins. Finally, genetic studies revealed that thismachinery is tightly linked to human diseases. Here, we describe these exciting findings as well as recent bio-chemical and cellular advances, and discuss possible impact on both human physiology and pathology.

© 2012 Elsevier B.V. All rights reserved.

1. Introduction

The ε-amino group of lysine residues can undergo different modifi-cations, including acetylation and other forms of acylation, sumoylation,biotinylation, ubiquitylation and methylation. Although extensivelystudied, the precise function(s) of the above modifications are stilllargely unknown. Lysine acetylation was originally identified morethan forty years ago on the N-terminal tail of histone proteins, whereits transient status regulates the interactionwith chromatin and, as a re-sult, DNA transcription [1]. Soon after, a large number of cytosolic andnuclear proteins were reported to be transiently acetylated in one ormore lysine residues. In addition to histones, they include proteins in-volved inDNA recombination and repair, transcription factors, cytoskel-etal proteins, chaperones, signaling proteins, and several metabolicenzymes [2,3]. Recognized functions of Nε-lysine acetylation includeregulation of activity, molecular stabilization and conformational as-sembly of a protein [4]. The acetylation of the ε amino group of a lysine

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residue (more typically referred to as lysine acetylation) differs fromNα-acetylation and O-acetylation. Nα-acetylation (also called N-terminalacetylation) occurs on the α-amino group at the N-terminus of nascentcytosolic proteins and is thought to be irreversible. It is mainly aco-translational event and predominantly affects alanine, serine and me-thionine residues; additionally it can also occur—albeit less often—on gly-cine, threonine, valine and cysteine residues [5]. O-acetylation wasdescribed on serine and threonine residues by two independent groupswhile studying the virulence of bacteria of the genus Yersinia [6,7]. Al-though the acetyltransferase activity of the bacterial effector Yersiniaouter protein J (YopJ) can target host proteins, an endogenous serine/threonineO-acetyltransferase activity has not been identified inmamma-lian cells. Both Nα- and O-acetylation are still poorly understood and willnot be discussed by this review.

From the biochemical perspective, lysine acetylation requires threeessential components: (1) an acceptor of the acetyl group (a proteinthat has the appropriate lysine residues); (2) a donor of the acetylgroup (acetyl-CoA); and (3) an enzyme able to transfer the acetyl groupfrom the donor to the acceptor (an acetyl-CoA:lysine acetyltransferaseor simply called acetyltransferase). The above three components wereinitially identifiedonly in the cytoplasmandnucleus; as a result, itwas as-sumed that lysine acetylation could only occur in the cytosol or in the nu-cleus [4,8]. However, in 2006 Schwer et al. reported the transient lysineacetylation of the mitochondrial matrix protein acetyl-CoA synthetase

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Box 1Acetyl-CoA biosynthesis in the cytosol.

The generation of Acetyl-CoA in the cytosol requires conversion ofpanthothenate (Vit B5) into Coenzyme A (CoA), a process that in-volves five enzymatic steps [117]. The first reaction, phosphoryla-tion of pantothenate to 4-phospho-pantothenate, appears to actas the rate-limiting step and is catalyzed by pantothenate kinase(Pank) (see Fig. 1). Mammals have four different genes encodingdifferent versions of Pank. PANK1 encodes Pank1α and Pankβ,both found exclusively in the cytosol; PANK2 encodes a mitochon-drial as well as at least one shorter cytosolic isoform (the catalyticactivity of two additional shorter isoforms has not been dem-onstrated); PANK3 and PANK4 encode one cytosolic proteineach (the catalytic signature of Pank4 has not been proven).Defects in Pank2 are associated with Hallervorden–Spatz syn-drome (HSS), also known as pantothenate kinase-associatedneurodegeneration (PKAN), an autosomal recessive neurode-generative disorder that affects the central nervous system[118]. Genetic disruption of the common PANK gene, fumble(fbl), in D. Melanogaster results in increased lethality as wellas infertility in the surviving animals [119], while disruptionof mouse Pank2 results in retinal degeneration and azoosper-mia [120]. The possible involvement of the other Pank pro-teins in human diseases is currently unknown.The two main enzymes responsible for the biosynthesis ofacetyl-CoA in the cytosol are the ATP-citrate lyase (Acly) andthe acetyl-CoA synthetase (AceCS) (see Fig. 1). Homozygousdisruption of Acly in the mouse is lethal while heterozygous dis-ruption is viable [121]. No information on the physiological aswell as pathological impact of AceCS is currently available;however, genetic disruption of AceCS2 (also known as ACSS1)which is exclusively found in the mitochondria, leads to defectsin thermoregulation and energy production as well as high de-gree of mortality [122].Mitochondria and peroxisome pathways that contribute to cy-tosolic acetyl-CoA are described in the text.

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[9], whereas in 2007 Costantini et al. reported the transient lysine acety-lation of the nascent endoplasmic reticulum (ER)-based form of themembrane protein β-site APP cleaving enzyme 1 (BACE1) [10]. Afterthese initialfindings, large-scale proteomic approaches reported that sev-eral proteins localized in the mitochondrial matrix undergo Nε-lysineacetylation [11,12]; their acetylation status regulates the metabolism ofthe cell in response to nutrient availability [13]. Similarly, many mem-brane and secreted proteins were reported to undergo transient lysineacetylation in the ER lumen [14–16]. Finally, high-scale proteomics alsoidentified ER-resident chaperones and enzymes to be Nε-lysine acetylat-ed in their luminal portion [12,16]. Therefore, what was once a cytosolicand nuclear event now appears to be an essential component of mito-chondria and ER functions as well.

2. A novel form of post-translational regulation in the ER

That Nε-lysine acetylation can occur in the lumen of the ER becameevident in 2007whenwe discovered that the ceramide-mediated regu-lation of BACE1 metabolism required transient acetylation of the na-scent protein in the ER [10]. BACE1 is a type I membrane protein; it issynthesized in the ER and then transported to the plasma membranealong the secretory pathway. During biosynthesis, the N-terminalectodomain faces the lumen of the ER while the short C-terminal tailfaces the cytosol. Since the short C-tail has one single lysine residuewe initially thought that, although it was the nascent ER-based formof the protein to be modified, the acetylation was still a cytoplasmicevent. However, biochemical assessment as well as mass spectrometryrevealed that themodified lysine residueswere all in the ectodomain ofthe protein. This finding posed an immediate biochemical challenge: inorder for the reaction to occur, both the donor (acetyl-CoA) and the en-zyme (acetyltransferase) of the reaction must be available in the lumenof the organellewhen BACE1 is synthesized. Subsequent efforts resultedin the identification of an ERmembrane acetyl-CoA transporter [15] andtwo ER-based acetyltransferases [17].

2.1. The transporter

Acetyl-CoA serves as the common donor of the acetyl group for thereaction of lysine acetylation (Box 1). Coenzyme A (CoA) is the carrierof the acetyl group, which is linked to the β-mercaptoethylamine endof CoA by a high-energy thioester bond. Acetyl-CoA originates fromthe break-down of carbohydrates, fatty acids and amino acids.Mamma-lian cells have three main and distinct pools of acetyl-CoA: a cytosolic/nuclear pool, amitochondrial pool, and a peroxisomal pool. The cytosol-ic and nuclear pools are here considered as one because acetyl-CoA canfreely pass through the nuclear pore complex [18]. A fourth and smallerpool of acetyl-CoA, which depends on active import from the cyto-sol, also exists in the ER and is essential for ER-based acetylation(see below). Because of its highly charged structure, acetyl-CoAcannot cross a lipid bilayer and is completely impermeable to cellmembranes. This obstacle is resolved by the existence of specificmembrane transporters that ensure continuous supply from boththe mitochondria and the peroxisomes.

Mitochondrial acetyl-CoA is transported to the cytosol as citrateby the tricarboxylate membrane transport system. Once in the cyto-sol, citrate is rapidly converted into oxaloacetate by the ATP-citratelyase (Acly) releasing acetyl-CoA (Fig. 1). The reaction requires ATPand free CoA. The oxaloacetate is then converted into pyruvate bythe sequential action of the malate dehydrogenase and malic enzymeand transported back into the mitochondria by the pyruvate mem-brane transporter. Here, pyruvate re-enters into the “pyruvate–citratecycle” ensuring continuous transfer of acetyl-CoA from the mitochon-dria to the cytosol [19]. A carnitine acetyltransferase-carnitine acetyltranslocase (CAT) system also appears to contribute to the activetransport of mitochondrial acetyl-CoA into the cytosol [20].

Peroxisomal acetyl-CoA is transported to the cytosol as part of differ-ent pathways, depending on the organism and cell type [21,22] (Fig. 1).The first involves conversion of acetyl-CoA into acetyl-carnitine by theperoxisomal enzyme carnitine acetyltransferase. Following export fromthe peroxisome, acetyl-carnitine can enter the mitochondria for furthermetabolism or feed into the cytosolic/nuclear pool of acetyl-CoA[21,23–25]. Peroxisomal acetyl-CoA can also enter the glyoxylatecycle to produce succinate, which is subsequently transported tothe cytosol and mitochondria for further metabolism [21]. Finally,acetyl-CoA can be hydrolyzed within the peroxisome to acetateand CoA; it has been suggested that this free acetate can be exportedto the cytosol where it is used to generate acetyl-CoA [22,26]. Themechanisms of acetyl-CoA transport from the peroxisomes to the cy-tosol are still poorly understood and the relative contribution of theabove pathways in mammals is currently uncertain.

In addition to the conversionof citrate byAcly, cytoplasmic acetyl-CoAalso originates from the condensation of free acetate and CoA byacetyl-CoA synthetase (AceCS) (Fig. 1). Both Acly and AceCS are alsopresent in the nucleus, suggesting that acetyl-CoA production can alsooccur in the nuclear compartment (independently from the cytosolicpool), where it regulates histone acetylation and, therefore, geneexpression [27].

The cytosolic pool of acetyl-CoA serves as the donor of the acetylgroup for the reaction of lysine acetylation occurring in the cytosol,

Fig. 1. Biochemical pathways that contribute to cytosolic Acetyl-CoA. Pathways as well as specific key enzymatic steps are described in the text and in Box 1.

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nucleus and ER lumen. As discussed above, the highly charged natureof acetyl-CoA impedes free passage across the lipid bilayer. Therefore,if lysine acetylation occurs in the lumen of the ER, a specific transport-er must exist in the ER membrane. Alternatively, enzymes responsiblefor acetyl-CoA synthesis could grant local production of the metabo-lite, assuming that its precursors are also available in the lumen ofthe ER. However, no evidence for a luminal synthesis of acetyl-CoAin the ER exists. To address this apparent biochemical obstacle, weinitially assessed whether highly purified ER vesicles were able to im-port free acetyl-CoA [10]. The experiments revealed that ER vesicleswere able to import acetyl-CoA in a concentration- and temperature-dependent fashion, suggesting a carrier-mediated process. Importantly,acetyl-CoA transport was found to be saturable with an apparent Km of14 μM[10], which is in the range of cytosolic acetyl-CoA availability andKm of other acetyl-CoA utilizing enzymes [28–30].

Interestingly, in 1997, ten years before we discovered that lysineacetylation occurs in the lumen of the ER [10], Kanamori et al. reportedthe identification of a putative acetyl-CoA transporter in the ER mem-brane [31]. The gene product, solute carrier family 33 member 1(SLC33A1)/Acetyl-CoA Transporter-1 (AT-1), was identified as part ofa screen for the Golgi-resident acetyltransferase responsible for theO-acetylation of complex gangliosides. Surprisingly, SLC33A1/AT-1(simply called AT-1 later on) displayed features that were consistentwith a putative ER membrane transporter and appeared to influencethe acetylation of non-lipid constituents rather than gangliosides. As aresult, the group led by Hirabayashi proposed a possible role of AT-1as an ERmembrane acetyl-CoA transporter [31,32]. Further biochemicalstudies from our laboratory confirmed that AT-1 was indeed the ERmembrane acetyl-CoA transporter (Fig. 2). Importantly, the functionalidentification of the transporter was achieved by biochemical reconsti-tution of the transport activity into artificial liposomes [15].

AT-1 is 549 amino acids long, migrates as a ~60–62-kDa band onreducing electrophoresis, and is predicted to have nine-to-twelve

transmembrane domains. Finally, it displays an exclusive ER localiza-tion, which is consistent with its biochemical function [15]. AT-1-mediated translocation of acetyl-CoA is inhibited with high specificitybyCoA, suggesting that the CoAmoiety serves as recognition signal [15].

2.2. The acetyl-CoA:lysine acetyltransferases

As with the transporter, the existence of an acetyl-CoA:lysineacetyltransferase activity in the lumen of the ER was first proven withclassical biochemistry. In fact, affinity purified BACE1 could be acetylat-ed in vitrowhen radiolabeled acetyl-CoAwas added togetherwith high-ly purified ER vesicles. Importantly, acetylation was only observed inthe presence of permeabilized ER vesicles, indicating that the enzymaticactivity resided in the lumen of the organelle [10]. The human genomewas then searched for genes encoding proteins that displayed structuralsimilarities to the catalytic domain of existing acetyltransferases.The analysis resulted in the identification of two different ER-basedacetyltransferases, which were named ATase1 (also known as camello-like 2 and N-acetyltransferase 8B) and ATase2 (also known as camello-like 1 and N-acetyltransferase 8) [17]. The two transferases are 86%identical at the protein level. Both have one short cytosolic tail, a sin-gle transmembrane segment and a larger endolumenal domain withthe catalytic activity (Fig. 2) [17]. When separated on gel electrophore-sis, they have an apparent molecular mass of ~28-kDa and ~25-kDa,respectively.

ATase1 and ATase2 are members of the camello family, which be-longs to the GNAT (Gcn5-related N-acetyltransferase) superfamily ofN-acetyltransferases. The GNAT superfamily includes over 10,000members grouped in more than 12 protein families with diverse cel-lular functions and substrates [33,34]. Members of this superfamilyshare the GNAT (acetyltransferase) domain, which contains four con-served sequence motifs, known as Motif C, D, A and B (ordered fromN-terminus to C-terminus) [33–35]. Although the homology at the

Fig. 2. Nε-lysine acetylation in the lumen of the ER. A, The ERmembrane transporter AT-1ensures a continuous supply of acetyl-CoA into the ER lumen while ER-membrane an-chored acetyltransferases (ATase1 and ATase2) catalyze the transfer of the acetyl groupto seven lysine residues of BACE1. B, Covalent modification of the Nε-amino group of a ly-sine residue by acetylation.


level of primary sequence, between different members of the super-family, might not be outstanding (3–23% pairwise sequence identity),the structure of the GNAT/acetyltransferase domain is highly con-served [34,35]. The GNAT conserved fold contains an N-terminalbeta strand (B1) followed by two alpha helices (H1 and H2), three an-tiparallel beta strands (B2–B4), a central alpha helix (H3), anotherbeta strand (B5), a fourth alpha helix (H4) and a final beta strand (B6)[34]. The lowest degree of conservation is found in the N-terminalmotif C (B1–H1) while the strongest is found in motifs D (B2–B3), A(B4–H3) and B (B5–H4). Together they make the HAT (histoneaceytltransferase) core (Fig. 3) [33,35,36]. Motif A is the most con-served motif from prokaryotes to eukaryotes and is also present inp300/CBP and MYST family (named after its founding members:MOZ, Ybf2/Sas3, Sas2 and Tip60) [37]. It contains the sequence Arg/Gln-X-X-Gly-X-Gly/Ala that is important for acetyl-CoA recognition andbinding, establishing most of the direct contacts to bound CoA. Remark-ably, in spite of the high degree of sequence identity, the alignment ofATase1 and ATase2 sequences revealed three non-conservative aminoacid substitutions inside theHAT core. Further studies are required to de-termine any functional consequences of these substitutions. Interesting-ly, two of these substitutions are located inside Motif A and one of themis in the sequence that makes up direct contact to bound CoA (Fig. 3).These differences may have important consequences for the kinetics ofthe enzymatic activity and, perhaps, could explain the high degree of

selectivity as well as the lack of off-site effects displayed by ATase1/ATase2 inhibitors [38].

ATase1 and ATase2 are differentially expressed in a variety of immor-talized as well as primary cells [38]. Both in themouse and human brain,they appear to bepreferentially—if not exclusively—expressed inneurons[38]. Finally, they are up-regulated in patients affected by Alzheimer'sdisease [38], further supporting a possible disease-association.

CML2 (the gene encoding ATase1) and CML1 (the gene encodingATase2) appear to be the result of a gene duplication event that oc-curred after the ape-Old World monkey split [39]. Although no diseaseassociation has been reported, several single nucleotide polymorphisms(SNPs) have been identified on both genes in the human population.Particularly, one SNP (accession no. rs4852974) on ATase1 generates astop codon in themiddle of the acetyl-CoA:lysine acetyltransferase cat-alytic domain and is predicted to inactivate the enzyme. The SNP seemsto be present in less than 2% of the general population.Whether this re-sults in decreased ATase activity is unknown. Also unknown is the bio-logical relevance of this apparent inactivation.

It is likely that ATase1 and ATase2 are not the only ER-basedacetyltransferases. Interestingly, calreticulin, an ER luminal proteinthat is mostly known as a Ca+2-binding chaperone, appears to haveacetyl-CoA:lysine acetyltransferase activity in vitro [40]. Whethercalreticulin retains acetyltransferase activity in vivo remains to bedetermined. Also to be determined are its potential substrates. However,considering the number of ER-resident and -transiting proteins that in-teract with calreticulin in the ER lumen, this potential acetyltransferaseactivity could have large impact.

In vitro assessment revealed that ATase1, but not ATase2, un-dergoes autoacetylation in one or more lysine residues [17]. The bio-logical significance of this different behavior is still unclear. In the case ofother acetyltransferases, the autoacetylation event has been describedeither as part of an allosteric mechanism that regulates the activity ofthe acetyltransferase or as part of the catalytic signature of thetransferase (see below in the Conclusive remarks and unresolvedquestions section). The allosteric switch is meant to regulate the activ-ity of the acetyltransferasewithout involving transcription/translation ofthe enzyme. In fact, when “hypo-acetylated” the enzyme displays re-duced activity, whilewhen “hyper/auto-acetylated” the enzyme displaysincreased activity [41,42]. This scenariowould be consistentwith the factthat ATase1 appears to act as a constitutive formof acetyltrasferasewhileATase2 appears to be tightly controlled at the transcriptional level [38].

2.3. The disposing machinery

To further study the role of lysine acetylation in the metabolism ofnascent BACE1, wemutated the seven lysine residues that are normallyacetylated in the native protein into alanine, arginine or glutamine. TheLys-to-Ala and Lys-to-Arg substitution generates “loss-of-acetylation”mutants whereas the Lys-to-Gln substitution generates a “gain-of-acetylation”mutant form of BACE1. The use of the above mutants re-vealed that the acetylated intermediates of BACE1 are able to reachthe Golgi apparatus and complete maturation while the non-acetylatedintermediates are retained in the ER Golgi Intermediate Compartment(ERGIC) [10]. Although efficiently synthesized in the ER and not able toreach the Golgi apparatus, the “loss-of-acetylation” mutants did not ap-pear to accumulate in the ERGIC or to be disposed of by the proteasomemachinery [10]. This observation suggested that an alternative pathwaywas responsible for the disposal of non-acetylated BACE1 intermediates.Subsequent studies revealed that proprotein convertase subtilisin kexintype 9 (PSCK9)/neural-apoptosis-regulated convertase 1 (NARC-1) wasan essential component of the disposing machinery [14] (Fig. 4).

PCSK9/NARC-1 is a member of the subtilisin (S8) family of serineproteases [43,44]. More specifically, PCSK9/NARC-1 is part of the S8Asubfamily, which so far includes only two members, PCSK9/NARC-1 it-self and site-1 protease (S1P), known to regulate the processing andconsequent activation of several ER-based transcription factors [45].

image of Fig.�2

Fig. 3. Alignment of the primary sequence of the ATases HAT core with prominent members of the GNAT superfamily. A, Sequence logo of the GNAT domain profile available atPROSITE (Database of protein domains, families and functional sites (http://prosite.expasy.org); PS51186). The sequence logo was generated after multiple sequence alignmentof 660 UniProtKB/Swiss-Prot true positive hits. The consensus sequence of the GNAT domain can be read from the top of the stacks. The height of each letter is proportional tothe frequency of the corresponding amino acid at the indicated position in the alignment. B, Primary sequence alignment of the HAT core highly conserved motifs of ATase1(NAT8B_HUMAN, Q9UHF3), ATase2 (NAT8_HUMAN, Q9UHE5) and other members of the GNAT superfamily, including Lysine acetyltransferase 2A also known as hGCN5(KAT2A_HUMAN, Q92830), Lysine acetyltransferase 2B also known as PCAF (KAT2B_HUMAN, Q92831) and aralkylamine N-acetyltransferase (AANAT) also known as SNAT(SNAT_HUMAN, Q16613). The first and last columns indicate the amino acid position from the corresponding full sequence. The numbers in parenthesis between the conservedmotifs indicate the length of the omitted sequence. The alignment was performed using the Clustal Omega multiple sequence alignment program (http://www.ebi.ac.uk/Tools/msa/clustalo). In red are highlighted fully conserved residues from the consensus sequence indicated in A. The presence of the second most frequently occurring consensusamino acid is indicated in yellow. The “:” (colon) indicates conservation between groups of strongly similar properties (scoring>0.5 in the Gonnet PAM 250 matrix) after alignmentof the ATase1 and ATase2 sequences with the GNAT consensus sequence. The “.” (period) indicates conservation between groups of weakly similar properties (scoring=b0.5 in theGonnet PAM 250 matrix) after alignment of the ATase1 and ATase2 sequences with the GNAT consensus sequence. The alignment of ATase1 and ATase2 sequences revealed thepresence of non-conservative amino acid substitutions in motifs A and D (arrows). The box in Motif A encloses the residues that have been shown to be important foracetyl-CoA recognition and binding in other members of the GNAT superfamily.


PCSK9/NARC-1 is a ~75-kDa proprotein that undergoes apparent auto-catalysis to generate a shorter form of ~65-kDa [46,47]. PCSK9/NARC-1has received great attention since the discovery that mutations in thePcsk9/Narc1 gene are associated with hypercholesterolemia. The factthat PCSK9/NARC-1 can regulate the degradation of the low-density li-poprotein (LDL) receptor (LDLR) has caused additional excitement aspossible down-regulation or biochemical inhibition of PCSK9/NARC-1might serve as an alternative approach for reducing hypercholes-terolemia. In fact, low levels or activity of PCSK9/NARC-1 wouldlead to increased expression of LDLR in the liver and increased uptake/degradation of circulating LDL [43,44].

Although the exact role of PCSK9/NARC-1 in LDLR metabolism is stilluncertain, early work showed that one of its essential functions is to reg-ulate the disposal of LDLR in a “post-ER compartment” while transitingalong the secretory pathway [48,49]. The “post-ER compartment”mech-anism was briefly put aside by evidence that PCSK9/NARC-1 could alsodegrade cell-surface LDLR following secretion in the conditioned media[50–52]. However, recent work has spurred new interest in the “post-ERcompartment”mechanism [53]. The fact that PCSK9/NARC-1 could affectthe levels of the nascent form of LDLR in a post-ER compartment whiletrafficking along the secretory pathway [48,49] stimulated our own in-terest. The possible involvement of PCSK9/NARC-1 in the disposal ofnon-acetylated intermediate forms of BACE1 in the ERGICwas addressedby a combination of biochemical andmolecular biological approaches. Inparticular, over-expression of PCSK9/NARC-1 in cultured cells decreasedthe levels of BACE1whereas siRNA-mediated down-regulation of PCSK9/NARC-1 achieved the opposite results [14]. Finally, the levels of BACE1were found to be increased in the brain of PCSK9/NARC-1 knockout ani-mals [14]. Importantly, down-regulation of PCSK9/NARC-1 increased thelevels of the “loss-of-acetylation”mutant forms of BACE1 but not those ofthe “gain-of-acetylation”mutants, suggesting that PCSK9/NARC-1 mightwork in concert with the ER-based acetylationmachinery to regulate thedisposal of BACE1 non-acetylated intermediates [14]. This conclusion isfurther supported by the observation that, similarly to BACE1, the

nascent ER-form of LDLR is acetylated [14], suggesting a common func-tion for the serine protease.

It is also worth mentioning that the promoter region of Pcsk9/Narc1contains a sterol regulatory element (SRE). As a result, the mRNA levelsof PCSK9/NARC-1 are up-regulated by SRE-binding proteins (SREBPs),ER membrane-based transcription factors that control both biosynthesisand uptake of cholesterol [54,55]. As a consequence, the expression ofPCSK9/NARC-1 is down-regulated by increased cellular cholesterol con-tent [56], and upregulated by HMG-CoA inhibitors (also known as“statins”), which reduce cholesterol content [57]. These results are par-ticularly important considering that several aspects of cholesterol me-tabolism have been associated with Alzheimer's disease [58], includingthe fact that increased cellular levels of cholesterol (which are predictedto decrease PCSK9/NARC-1-mediated disposal of BACE1) increase Aβgeneration, whereas reduced levels of cholesterol (which are predictedto increase PCSK9/NARC-1-mediated disposal of BACE1) reduce Aβgeneration.

Although initially studied for its implication in LDLR metabolism,it is now evident that PCSK9/NARC-1 “does”more than just regulatingLDLR levels. In fact, in addition to BACE1 [14] and APP [15], it also reg-ulates the disposal of other cell-surface membrane proteins [59,60]and perhaps secreted proteins as well [61], suggesting a more globalfunction. Indeed, a recent proteomic study identified more than 300proteins that are influenced by PCSK9/NARC-1 activity [62]. The listincludes cell-surface proteins as well as intracellular proteins, proteinsinvolved in folding and post-translational modification in the early se-cretory pathway, proteins involved in vesicle and organelle transport,cytoskeleton as well as cell-adhesion proteins. This global impact ofPCSK9/NARC-1 was also demonstrated by two microarrays studies,which implicated this serine protease in many biological pathways[63,64].

We initially reported that inhibition of the proteasome machinerydid not appear to affect the disposal of the “loss-of-acetylation”mutantforms of BACE1 [10]. However, recently Gong et al. have proposed the

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http://prosite.expasy.org

http://www.ebi.ac.uk/Tools/msa/clustalo

http://www.ebi.ac.uk/Tools/msa/clustalo

Fig. 4. Schematic view of BACE1 acetylation. Nascent BACE1 is transiently acetylated in the lumen of the ER by ATase1 and ATase2. If acetylated, nascent BACE1 can reach the Golgiapparatus and complete maturation. In contrast, non-acetylated BACE1 is retained and degraded in the ERGIC. The ER-based acetylation also requires translocation of acetylCoAfrom the cytosol into the lumen of the ER by the ER membrane transporter AT-1. The acetyl group is shown as a red circle.


possible requirement of the E3-ligase activity of the Fbx2-SCF ubiquitinligase complex, suggesting that the ubiquitin–proteasome systemmightstill be involved with the clearance of non-acetylated species of BACE1[65]. The ubiquitylation of BACE1 is somewhat specific and seems tooccur on some of the seven lysine residues that are acetylated [65]; thiscould potentially explain why acetylated BACE1 intermediates are resis-tant to proteasome degradation [10]. Whether there is cross-talk be-tween PCSK9/NARC-1 and SCFFbx2-E3 ligase remains to be assessed. Infact, PCSK9/NARC-1 is a serine protease aswell as a chaperone-like pro-tein, while Fbx2 is typically involved in the recognition and binding ofhigh-mannose oligosaccharides onmisfolded/unfolded ER luminal pro-teins, which are subsequently targeted for proteasome-dependent dis-posal (see also later). Another issue that will also need furtherinvestigation is how the Fbx2-SCF ubiquitin ligase complex can have ac-cess to the ectodomain of BACE1; in fact, the former is in the cytosolwhile the latter is in the lumen of the ER. It is likely that nascentnon-acetylated BACE1 might need to be “pre-processed” and retro-translocated to the cytosol before becoming a substrate of the Fbx2-SCFubiquitin ligase complex, as described for other Fbx2-SCF substrates [66].

3. A novel mechanism for quality control and/or ERAD?

The essential information for the correct assembly of membraneand secreted proteins synthesized in the ER is already present intheir primary amino acid sequence. However, the efficiency of foldingitself is ensured and enhanced by ER-resident proteins that act aschaperones and/or modifying enzymes. Chaperones assist in the fold-ing by ensuring safe passage of the nascent protein chain through theER membrane, by retrieving and preventing aggregation of incorrect-ly folded protein intermediates, and—in some cases—by protecting

the nascent polypeptide from interacting with potential ligands inthe ER [67,68]. Chaperones can be solely restricted to the ER, can travelthrough the secretory pathway together with their substrates or, insome cases, be secreted to the extracellularmilieuwith their immediatesubstrates [67–69]. The large family of ER chaperones includes heat-shock proteins such as BiP, calreticulin, calnexin and the receptor asso-ciated protein (RAP). In contrast to chaperones, modifying enzymestypically ensure correct folding by completing the information alreadycontained in the primary amino acid sequence. Some examples arethe protein disulfide isomerase (PDI), which catalyzes the oxidation offree SH groups of paired cysteine residues to form disulfide bonds(-CH2\S\S\CH2-), and the oligosaccharyltransferase (OST), whichtransfers a preformed “core” oligosaccharide from a glycolipid donor,the dolichol-P-P-(GlcNAc)2(Man)5(Glc)3, to an asparagine residue inthe consensus sequence Asn-X-Ser/Thr of the nascent glycoprotein. Al-though the above are examples of permanent covalent modifications,there are also transient modifications that are only meant to recognizeand salvage incorrectly folded protein intermediates. This is the caseof the UDP-glucose:glycoprotein glucosyltransferase (UGGT), which at-taches one glucose residue to improperly folded nascent glycoproteinsand regulates its interaction with the lectin chaperone calnexin [70,71].

The above chaperones andmodifying enzymes not only ensure fidel-ity of the “protein code” by assisting nascent polypeptides in assumingthe right conformation, which is necessary to their function, but also se-quester incorrectly folded intermediates that have failed quality control(QC). These are generally retained in the ER and directed toward theER associated degradation (ERAD). Incorrectly folded polypeptides thatare disposed of by the ERAD are typically retro-translocated to the cyto-sol and sent to the proteasome for disposal but can also aggregate (in thecytosol or in the ER itself) to form protein inclusions. The fate of the

image of Fig.�4


aggregates is uncertain; they can serve as a “sink” for misfolded proteinsthat failed QC, thus preventing their otherwise toxic functions (thisseems to be the case for the cytoplasmic aggregates called “aggresomes”)or can be digested through autophagic-like mechanisms (see below)[68,72–76]. However, under certain conditions, protein aggregates canbe overwhelming for the cell and become toxic themselves. Misfoldedprotein intermediates can also bypass the above more classical ERADand be directed to the Golgi apparatuswhere they are retained or direct-ed to the lysosomes for ultimate degradation [68].

3.1. To protect the good ones

The use of the acetylationmutants revealed that the acetylated inter-mediates of BACE1 are able to reach the Golgi apparatus and completematuration while the non-acetylated intermediates are retained in theERGIC [10]. In addition, the down-regulation of PCSK9/NARC-1 only in-creased the levels of the “loss-of-acetylation” mutant, suggesting thatsome sort of selection takes place [14]. These features are reminiscentof a QC event where certain biosynthetic intermediates are selected for“free passage” across the secretory pathway while others (presumablyunfolded/misfolded intermediates) are not (see Fig. 4). Although thepossible connection between lysine acetylation in the ER and QC stillneeds to be carefully evaluated, this possibility is supported by the“chaperone-like” activity of the acetyltransferases [17]. Perhaps, theATases can only recognize and acetylate correctly folded intermediates.Perhaps, the Nε-lysine acetylation provides away to “lock” the acetylated(correctly folded) intermediates to the transferases protecting themfrom PCSK9/NARC-1 mediated disposal. The locking mechanisms wouldexplain why all seven acetylated lysine residues in BACE1 are locatedon the same face of the protein [10]. Perhaps, the release of the ATases[17] togetherwith the deacetylation of BACE1 that occurs in the Golgi ap-paratus [10] serves to “restore” the original “protein code” allowing finalmaturation and transport to the cell surface. Perhaps, the ATases do notsimply serve to recognize already folded protein intermediates but ac-tively participate in the folding itself. In the case of BACE1, the acetylatedlysine residues are clustered in areas that are characterized by low-electron density (and intrinsic conformational flexibility). This structuralflexibility appears to be essential for the catalytic activity [77–79] as wellas for the natural “shedding” [80] of the enzyme. It is possible that theNε-lysine acetylation—by neutralizing the positive charges of the lysineresidues—serves to stabilize and facilitate the folding of kinetically unfa-vorable areas of the nascent protein [10]. Perhaps, the “stabilization” and“locking” mechanisms delineated above are intimately connected toachieve greater efficiency while processing large quantities of nascentmembrane and secreted proteins in the ER. Although we recognize thatthe above “perhaps” still need to be biochemically proven, our current in-formation appears to preclude other possibilities. This view is alsosupported by the fact that BACE1 is not alone; in fact, other membraneproteins undergo the same transient post-translational modification[14–16].

3.2. What if it fails?

If the ER-basedNε-lysine acetylationmachinery is an essential formofpost-translational regulation of nascent proteins, we would expect theglobal failure of the ER-based acetylation to lead to cellular disaster. Thedown-regulation of the two transferases, ATase1 and ATase2, did not re-sult in significant cell toxicity [17]. However, it is likely that the ER hasmore than just two acetyltransferases (discussed above); it is also likelythat the transferases share (at least in part) their substrates allowingfor one enzyme to compensate for the lack of the other(s). In contrastto theATases, the down-regulation of AT-1 resulted inmassive andwide-spread cell death, indicating that the translocation of acetyl-CoA into theER lumen is an essential cellular function [15,81]. This conclusion isstrongly supported by the fact that AT-1 is mutated in patients affectedby autosomal dominant spastic paraplegia-42 (SPG42) [82].

SPG42 is part of the highly heterogeneous family of hereditary spasticparaplegias (here collectively called SPGs). The clinical heterogeneityprobably reflects the genetic complexity of the disease. A recent list ofgenes and loci implicated with SPGs has been reported by Salinas et al.[83]. SPGs are generally divided into two groups: uncomplicated andcomplicated. Uncomplicated SPGs are typically characterized byprogres-sive bilateral spasticity and peripheral neuropathy; urinary symptomsandmild cognitive decline are also present in a large number of patients.The onset of the disease varies greatly from early childhood to as late as70 years of age. Themain pathological feature of uncomplicated SPGs is aprogressive degeneration of motor axons of the corticospinal tract in theabsence of evident muscle pathology. Loss of volume of the corpuscallosum andmemory-forming areas of the brain, as well as disseminat-ed lesions of cerebral white matter, have also been reported [83,84].Complicated SPGs are characterized by the fact that the lower limb spas-ticity is accompanied by other features such as severe mental retarda-tion, dementia, epilepsy, cerebellar ataxia, amyotrophy, retinopathy,glaucoma, deafness and icthyosis [83,84].

Enlarged ER sheets and tubules as well as generalized defects of theER network constitute a commonmorphological feature of SPG neurons(reviewed in [85]). Importantly, the vast majority of mutations found inSPGs are concentrated in genes encoding proteins that are localized inthe ER and ERGIC, function at the ER-to-Golgi interface or are overall in-volved in ER network organization, membrane trafficking, and axonaltransport of macromolecules and other cargoes (reviewed in [83,85]).This finding should be viewed together with the fact that the earliestandmost striking changes observed in SPG patients are always concen-trated in the long axons of descending and ascending spinal neurons. Inhumans, these processes canmeasure up to onemeter in length. As a re-sult of their morphology, they are heavily dependent on membranetrafficking, rapid axonal transport of cargo molecules from the ER tothe plasma membrane and active cytoskeletal organization. A defectin the above events is likely to disrupt their function and lead to degen-eration. Interestingly, haploinsufficiency or leaky mutations associatedwith SPGs only cause defects/degeneration of neurons, further stressingthe strong dependence of neuronal cells to the above events [86,87]. Al-though ER network organization, membrane trafficking, and transportof macromolecules are essential and common features of every cell,neurons are simplymore dependent on them. However, less dependencedoes not mean no dependence! In fact, a ~95% decrease in AT-1 levelscausedmassive death of cultured cells under ex vivo conditions [15]. Fur-thermore, down-regulation of AT-1 in the zebrafish increased embryoniclethality and caused a curved-shaped tail phenotype in the surviving an-imals, as well as defective axonal growth of spinal cord neurons [82]. Thestriking phenotype caused by the down-regulation of AT-1 under both exvivo [15] and in vivo [82] conditions indicates that a constant influx ofacetyl-CoA into the lumen of the ER is an essential cellular function(discussed later).

Mutations in AT-1 have also been identified in five patients affectedby a complex autosomal-recessive syndrome characterized by psycho-motor retardation and severe developmental delay. The patients, whodisplayed brain atrophy, cerebellar hypoplasia, hypomyelination, con-genital chataracts, hearing loss, andmulti-organ failure, died premature-ly within the first 5 years of their life [88]. In contrast to SPG42 patients,whowere heterozygous for themutation [82], the abovewere all homo-zygous [88]. The above patients also displayed low serum copper and ce-ruloplasmin [88]. However, reassessment of one of the above fivepatients identified an additional mutation in the copper chaperone forsuperoxide dismutase (CCS) [89], suggesting that the phenotype mightbe complicated by the co-presence of more than one mutation.

3.3. Regulation of autophagy/ERAD(II) by the ER-based acetylationmachinery

A close assessment of themodalities of the cell death associatedwiththe down-regulation of AT-1 in culture revealed that cells initially


respond to the reduced import of acetyl-CoA into the ER lumen by in-creasing the size of the ER tubules [15]. This is then followed by the for-mation of autophagosomes in close proximity of the ER membrane,suggesting that the ER membrane itself participates in the formationof the autophagic elements. If the down-regulation of AT-1 persists,the entire ER breaks down into autophagosomes and ultimately thecell dies by autophagic cell death [15]. Importantly, before widespreaddigestion of intracellular organelles (final autophagic cell death) theER is the only organelle that appears to be affected. The above observa-tion clearly indicates that the continuous supply of acetyl-CoA into theER lumen is an essential feature of this organelle; they also indicatethat autophagic digestion of ER tubules is the preferential strategy todeal with the failure of the ER-based acetylation machinery.

Autophagy is a cellular process that allows turnover and/or degrada-tion of subcellular components, which are initially engulfed in double-membrane structures and then diverted toward active lysosomes forfinal degradation. This process can be part of typical “house-keeping”activities that dispose of unwanted proteins or excessive membranes;it can also serve to ensure supply of nutrients during starvation or toclear the organism of unwanted cells during development [76,90–93].As described above, the ER has mechanisms in place that ensure correctfolding of secreted/membrane proteins. When these fail, unwanted(unfolded/misfolded) proteins are typically degraded by the proteasomeas part of ERAD. However, the proteasome preferentially degradesmono-meric proteins that are unfoldedprior to retro-translocation across the ERmembrane [68]. Large protein aggregates are mostly dealt with byexpanding the ER and activating autophagy. The autophagic process al-lows the cell to counterbalance the ER expansion and dispose of the pro-tein aggregates at the same time [73–75,94]. This process is still part ofERAD and the terms ERAD(I) and ERAD(II) have also been used to differ-entiate the ubiquitin/proteasome process from the autophagic process,respectively [95]. Therefore, autophagy must be seen as an essentialcellular function that ensures disposal of unwanted material. This is par-ticularly important for neurons where a high threshold for ER stress(discussed above) and inherent cellular features require a continuousand efficient way to dispose of protein aggregates [92,96–99]. Obviously,if unchecked, autophagy can become terminal (autophagic cell death;also referred to as type II cell death to differentiate it from apoptosis,which is referred to as type I cell death). Both ERAD(I) and ERAD(II) areunder the control of the unfolded protein response (UPR), a complexER-based signaling machinery that corrects possible imbalances that re-sult in ER stress and adjusts the ability of the organelle to cope with theaccumulation of unfolded/misfolded protein intermediates [68,100–102].

That the ER-based acetylation machinery is intimately linked to theUPR and ERAD(II) in the ER is supported by four main recent findings.First, a large number of ER-resident proteins involved with folding anddisposal of unfolded/misfolded protein intermediates is acetylated inthe lumen of the ER [16]. Second, already known and well characterizedER stress inducers increase the influx of acetyl-CoA into the ER lumen byup-regulating the expression of AT-1 [81]. Third, the up-regulation ofAT-1 during ER stress was linked to one of the three main branches oftheUPR, specifically to the inositol-requiring protein-1 (IRE1)-dependentsignaling pathway [81]. IRE1 is an ER-membrane bound protein thatwhenactivated, as a result of ER stress, removes a small fragment (intron)from the mRNA of X-box binding-1 (XBP1). The process requires theendoribonuclease activity of IRE1 and the ligase activity of an unknownligase [102,103]. The product of the spliced version of XBP1 (XBP1s) is apotent transcriptional activator of UPR target genes that promote ERADof unfolded/misfolded protein intermediates and ER biogenesis [104].Fourth, AT-1 was shown to regulate the activation of autophagy down-stream of XBP1 itself [81]. Therefore, the influx of acetyl-CoA into theER lumen is a novel and essential feature of the UPR and appears to con-trol the ability of the ER to recognize and select correctly folded proteinintermediates (as observed with BACE1), and dispose of unfolded/misfolded protein aggregates through the regulation of autophagy/ERAD(II). At the mechanistic level, the regulation of ERAD(II)

involves intraluminal acetylation of the autophagy protein Atg9A[81], which might act as an ER-based “sensor” for the acetylation statusof the ER (Fig. 5). Indeed, acetylation of Atg9A prevents the induction ofautophagy whereas the deacetylation (or lack of acetylation) has theopposite effect [81]. A similar regulatory function of the Nε-lysineacetylation has also been observed with the cytosolic members ofthe autophagic machinery, specifically Atg5, Atg7, Atg8 and Atg12[105,106].

It must be noted that the activation of the “ER stress signaling” is botha physiologic and pathologic event. Cells can experience transient pe-riods of ER stress as part of their activity [107–110]. As discussedabove, due to their specific functions, neurons may have to cope with asubstantial physiological burden and, as a result, have higher basal levelsof ER stress. This could potentially explain why amutation in AT-1 is pri-marily associated with a disease of the nervous system. However, B cellsalso experience reorganization and expansion of the ER and activation ofthe UPR when they differentiate into immunoglobulin secreting plasmacells [111]. The ultimate purpose is to ensure the biosynthesis and secre-tion of a large quantity of correctly folded immunoglobulins [111,112].Interestingly, the differentiation process of B cells into plasma cells isalso accompanied by activation of XBP1 signaling and up-regulation ofAT-1 [113]. Therefore, AT-1 does not only respond to ER stress-inducingexperimental drugs, which are largely non-physiologic, but also to“naturally” occurring conditions that require induction of the ERstress machinery.

4. Conclusive remarks and unresolved questions

Although “young”, the ER-based acetylation machinery is rapidlyemerging as a major component of normal ER functions and is poisedto grow in importance aswe further delineate its impact on physiologicand pathologic events. As we learn more, several currently unresolvedquestions will need to be addressed. For example:

What is the purpose of the ER-based acetylation? Studies performedwith BACE1 indicate that the Nε-lysine acetylation of the nascentprotein might serve to distinguish between correctly folded andunfolded/misfolded protein intermediates. Although it needs tobe proven, a similar mechanism might be in place for otherER-transiting proteins, which need to move out of the ER alongthe secretory pathway. However, why acetylate ER-residentproteins, which are not supposed to leave the organelle? Is there asimilar mechanism in place for ER-resident proteins? Perhaps, in thecase of ER-resident proteins the acetylation influences activity/function of the proteins rather than their ability to leave the organ-elle. It is also possible that lysine acetylation is necessary for theassembly of kinetically unfavorable (disordered?) regions of the na-scent polypeptide. This scenario could be tested by analyzingthe structural signature—when available—of recently identifiedER-resident and -transiting proteins that undergo acetylation [16].Finally, it is highly possible that the cell might have more than onereason to acetylate nascent proteins in the ER lumen.How many ATases are in the ER? At this point, it is evident that theER has more than two acetyltransferases. Although some overlap ispossible, it is likely that they have different substrates. Identificationof the other ATases aswell as their immediate substrateswill help ustarget the acetylation machinery for therapeutic purposes. ATase1and ATase2 are attractive targets for Alzheimer's disease therapeu-tics [17,38]. It is likely that other ATases might represent viable tar-gets for other diseases.What is the catalytic signature of the ATases? Most of the knownacetyltransferases employ two different catalytic mechanisms: theternary complex and the ping-pong mechanism [114] (Fig. 6). The

Fig. 5. Regulation of the influx of acetyl-CoA into the ER lumen as part of the UPR. The constant influx of acetyl-CoA into the ER lumen is required to control the induction ofautophagy/ERAD(II) as part of the UPR. Specifically, AT-1 acts down-stream of IRE1/XBP1 signaling. If the influx of acetyl-CoA is maintained, ERAD(II) helps to eliminate unwantedunfolded/misfolded aggregates without inducing cell death (A). However, if the influx is not maintained, programmed Type II (autophagic) cell death is activated (B). Theautophagy protein Atg9A seems to act as a “sensor” for the acetylation status of the ER.


ternary complex mechanism involves direct transfer of the acetylgroup from the donor (acetyl-CoA) to the acceptor lysine residue.This process requires the enzyme to interact and bind both thedonor and the acceptor at the same time (hence, ternary complex).In contrast, the ping-pongmechanism involves initial transfer of theacetyl group from the donor (acetyl-CoA) to the enzyme, which be-comes acetylated. The intermediate acetylated enzyme will thentransfer the acetyl group to the acceptor lysine residue. Therefore,this process requires the acetyltransferase to interact separatelywith the donor and the acceptor and to act as an acetylated interme-diate species (hence, ping-pong). These mechanistic differences arevery important because different mechanisms imply different clas-ses of inhibitors and different forms of regulation [114].If there are acetyltransferases, are there deacetylases as well? Ourinitial studies with BACE1 indicated that the nascent protein isnormally acetylated in the lumen of the ER and then deacetylatedin the lumen of the Golgi apparatus [10]. Indeed, in vitro biochemicalassessment revealed that the Golgi apparatus possess deacetylase ac-tivity [10]. Importantly, the activity was found to reside in the lumenof the organelle [10]. However, computer-based searches for possiblecandidates have—so far—failed to yield solid candidates, suggestingthat “more traditional” protein purification approaches will be re-quired to ensure the identification of another piece of this puzzle.Similar strategiesmight also be necessary to determinehow transientacetylation/deacetylation is achieved for ER-resident proteins.

Can AT-1 be targeted for therapeutic purposes? Initial studies indi-cate that AT-1 is essential for cell viability [15,82]. Indeed, down-regulation of AT-1 in mammalian cells triggers the activation ofthe autophagic machinery and results in widespread type I(autophagic) cell death [15,81]. As such, we should predictAT-1 not to be a viable target. However, what is toxic for normalcells might not be as toxic for sick cells. Indeed, mice expressinga mutant form of the Cu,Zn superoxide dismutase (SOD1) linkedto amyotrophic lateral sclerosis (ALS) benefit from an excessiveactivation of the autophagic machinery [115,116]. This appearsto be due to a more efficient removal of toxic protein aggregates.Perhaps, a balance between the rate of protein aggregates for-mation and the rate of autophagy-mediated removal of the ag-gregates must be ensured. As such, excessive autophagy wouldbe deleterious in a normal cell with normal levels of protein ag-gregates, but beneficial in a “sick” cell with excessive formation/accumulation of protein aggregates. Importantly, the protectiveeffect of a “hyper-active” autophagic machinery in the aboveALS-like model was achieved by down-regulating XBP1 [115,116],which appears to act through AT-1 itself [81]. Therefore, AT-1might be a possible target for therapeutics, albeit only in a selectedgroup of patients.How is the cross-talk between AT-1 and the autophagic machineryensured? Recent results indicate that the influx of acetyl-CoA intothe ER lumen is “sensed” by Atg9A, the only membrane bound

image of Fig.�5

Fig. 6. Schematic view of the two main catalytic mechanisms employed by acetyl-CoA:lysine acetyltransferases. Description is in the text.


autophagy protein [81]. However, the molecular details are still un-known. Perhaps the acetylation status of Atg9A regulates its traffick-ing to the Golgi apparatus and the consequent formation of the“autophagic membrane”. Or, perhaps, the acetylation status ofAtg9A affects its ability to interact with the other elements of theautophagic machinery.Is the proteasome machinery involved? How can we reconcile thefact that the disposal of non-acetylated BACE1 mutants is not affect-ed by proteasome inhibitors [10] with the fact that the lysine resi-dues that are acetylated appear to be necessary for the in vitroubiquitylation of BACE1 by the Fbx2-SCF ubiquitin ligase complex[65]? Are these two mechanisms related? Or are they meant to rec-ognize different intermediate species of the nascent protein?

Although many questions remain to be answered, one thing iscertain: what was once a cytosolic and nuclear event is now an essen-tial component of ER functions. Dissection of the biochemistry thatgoverns the ER-based acetylation machinery will certain lead us to abetter understanding of fundamental biological events that influencehuman physiology and pathology.

Acknowledgements

The authors wish to thank Dr. Rozalyn Anderson for critical read-ing of an early version of this manuscript. Luigi Puglielli is supportedby grants from the NIH/NIA (AG028569 and AG033514) and the De-partment of Veterans Affairs (Merit Award). Mariana Pehar is partial-ly supported by NIH (8P20GM103542).

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